RESUMEN
Tetratricopeptide repeat protein 37 (TTC37) is a causative gene of trichohepatoenteric syndrome (THES). However, little is known about the pathogenesis of this disease. Here, we characterize the phenotype of a Drosophila model in which ski3, a homolog of TTC37, is disrupted. The mutant flies are pupal lethal, and the pupal lethality is partially rescued by transgenic expression of wild-type ski3 or human TTC37. The mutant larvae show growth retardation, heart arrhythmia, triacylglycerol accumulation, and aberrant metabolism of glycolysis and the TCA cycle. Moreover, mitochondrial membrane potential and respiratory chain complex activities are significantly reduced in the mutants. Our results demonstrate that ski3 deficiency causes mitochondrial dysfunction, which may underlie the pathogenesis of THES.
RESUMEN
The synaptonemal complex (SC) is a conserved meiotic structure that regulates the repair of double-strand breaks (DSBs) into crossovers or gene conversions. The removal of any central-region SC component, such as the Drosophila melanogaster transverse filament protein C(3)G, causes a complete loss of SC structure and crossovers. To better understand the role of the SC in meiosis, we used CRISPR/Cas9 to construct 3 in-frame deletions within the predicted coiled-coil region of the C(3)G protein. Since these 3 deletion mutations disrupt SC maintenance at different times during pachytene and exhibit distinct defects in key meiotic processes, they allow us to define the stages of pachytene when the SC is necessary for homolog pairing and recombination during pachytene. Our studies demonstrate that the X chromosome and the autosomes display substantially different defects in pairing and recombination when SC structure is disrupted, suggesting that the X chromosome is potentially regulated differently from the autosomes.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Fase Paquiteno/genética , Complejo Sinaptonémico/genética , Cromosoma X/genética , Animales , Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Recombinación Genética/genética , Eliminación de Secuencia/genéticaRESUMEN
Succinyl-CoA synthetase/ligase (SCS) is a mitochondrial enzyme that catalyzes the reversible process from succinyl-CoA to succinate and free coenzyme A in TCA cycle. SCS deficiencies are implicated in mitochondrial hepatoencephalomyopathy in humans. To investigate the impact of SCS deficiencies in Drosophila, we generated a null mutation in Scs alpha subunit (Scsα) using the CRISPR/Cas9 system, and characterized their phenotype. We found that the Drosophila SCS deficiency, designated ScsαKO, contained a high level of succinyl-CoA, a substrate for the enzyme, and altered levels of various metabolites in TCA cycle and glycolysis, indicating that the energy metabolism was impaired. Unlike SCSα deficiencies in humans, there was no reduction in lifespan, indicating that Scsα is not critical for viability in Drosophila. However, they showed developmental delays, locomotor activity defects, and reduced survival under starvation. We also found that glycogen breakdown occurred during development, suggesting that the mutant flies were unable to produce sufficient energy to promote normal growth. These results suggested that SCSα is essential for proper energy metabolism in Drosophila. The ScsαKO flies should be useful as a model to understand the physiological role of SCSα as well as the pathophysiology of SCSα deficiency.
Asunto(s)
Acilcoenzima A/deficiencia , Proteínas de Drosophila/deficiencia , Drosophila melanogaster/fisiología , Metabolismo Energético , Privación de Alimentos , Locomoción , Acilcoenzima A/genética , Animales , Animales Modificados Genéticamente , Conducta Animal , Supervivencia Celular , Ciclo del Ácido Cítrico , Proteínas de Drosophila/genética , Drosophila melanogaster/enzimología , Drosophila melanogaster/genética , Glucógeno/metabolismo , Glucólisis , Masculino , Mitocondrias/metabolismo , Fenotipo , InaniciónRESUMEN
Egg activation is the process by which a mature oocyte becomes capable of supporting embryo development. In vertebrates and echinoderms, activation is induced by fertilization. Molecules introduced into the egg by the sperm trigger progressive release of intracellular calcium stores in the oocyte. Calcium wave(s) spread through the oocyte and induce completion of meiosis, new macromolecular synthesis, and modification of the vitelline envelope to prevent polyspermy. However, arthropod eggs activate without fertilization: in the insects examined, eggs activate as they move through the female's reproductive tract. Here, we show that a calcium wave is, nevertheless, characteristic of egg activation in Drosophila. This calcium rise requires influx of calcium from the external environment and is induced as the egg is ovulated. Pressure on the oocyte (or swelling by the oocyte) can induce a calcium rise through the action of mechanosensitive ion channels. Visualization of calcium fluxes in activating eggs in oviducts shows a wave of increased calcium initiating at one or both oocyte poles and spreading across the oocyte. In vitro, waves also spread inward from oocyte pole(s). Wave propagation requires the IP3 system. Thus, although a fertilizing sperm is not necessary for egg activation in Drosophila, the characteristic of increased cytosolic calcium levels spreading through the egg is conserved. Because many downstream signaling effectors are conserved in Drosophila, this system offers the unique perspective of egg activation events due solely to maternal components.
Asunto(s)
Calcio/metabolismo , Drosophila/metabolismo , Oocitos/metabolismo , Animales , Drosophila/citología , Inositol 1,4,5-Trifosfato/metabolismo , Transporte IónicoRESUMEN
Although traditional genetic assays have characterized the pattern of crossing over across the genome in Drosophila melanogaster, these assays could not precisely define the location of crossovers. Even less is known about the frequency and distribution of noncrossover gene conversion events. To assess the specific number and positions of both meiotic gene conversion and crossover events, we sequenced the genomes of male progeny from females heterozygous for 93,538 X chromosomal single-nucleotide and InDel polymorphisms. From the analysis of the 30 F1 hemizygous X chromosomes, we detected 15 crossover and 5 noncrossover gene conversion events. Taking into account the nonuniform distribution of polymorphism along the chromosome arm, we estimate that most oocytes experience 1 crossover event and 1.6 gene conversion events per X chromosome pair per meiosis. An extrapolation to the entire genome would predict approximately 5 crossover events and 8.6 conversion events per meiosis. Mean gene conversion tract lengths were estimated to be 476 base pairs, yielding a per nucleotide conversion rate of 0.86 × 10(-5) per meiosis. Both of these values are consistent with estimates of conversion frequency and tract length obtained from studies of rosy, the only gene for which gene conversion has been studied extensively in Drosophila. Motif-enrichment analysis revealed a GTGGAAA motif that was enriched near crossovers but not near gene conversions. The low-complexity and frequent occurrence of this motif may in part explain why, in contrast to mammalian systems, no meiotic crossover hotspots have been found in Drosophila.
RESUMEN
The Ca(2+)/Calmodulin-dependent phosphatase calcineurin is essential for exit from meiotic arrest at metaphases I and II in Drosophila and Xenopus oocytes. We previously found that Sarah, the Drosophila homolog of regulator of calcineurin, acts as a positive regulator of calcineurin and is required to complete anaphase I of female meiosis. Here, we undertook biochemical approaches, including MS and posttranslational modification analyses, to better understand the mechanism by which Sarah regulates calcineurin. A search for phosphorylated residues revealed that Sarah is highly phosphorylated at Ser100, Thr102, and Ser219 in both ovaries and activated eggs and that Ser215 is phosphorylated only in activated eggs. Functional analyses using mutant forms of Sarah showed that phosphorylation at Ser215, a consensus phosphorylation site for glycogen synthase kinase 3ß (GSK-3ß) and its priming kinase site Ser219, are essential for Sarah function. Furthermore, germ-line clones homozygous for a null allele of shaggy (Drosophila GSK-3ß) both fail to complete meiosis and lack phosphorylation of Sarah at Ser215, suggesting that the phosphorylation of Sarah by Shaggy/GSK-3ß is required to complete meiosis. Our findings suggest a mechanism in which Shaggy/GSK-3ß activates calcineurin through Sarah phosphorylation on egg activation in Drosophila.
Asunto(s)
Calcineurina/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiología , Drosophila/citología , Glucógeno Sintasa Quinasa 3/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Meiosis/fisiología , Alelos , Animales , Western Blotting , Proteínas de Unión al Calcio , Femenino , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Homocigoto , Inmunoprecipitación , Ovario/metabolismo , Óvulo/metabolismo , FosforilaciónRESUMEN
The pairing of homologous chromosomes and the intimate synapsis of the paired homologs by the synaptonemal complex (SC) are essential for subsequent meiotic processes including recombination and chromosome segregation. Here we show that the centromere clustering plays an important role in initiating homolog synapsis during meiosis in Drosophila females. Although centromeres are not clustered prior to the onset of meiosis, all four pairs of centromeres are actively clustered into one or two masses during early meiotic prophase. Within the 16-cell cyst, centromeric clustering appears to define the first step in the initiation of synapsis. Clustering is restricted to the nuclei that form the SC and is dependent on all known SC proteins. Surprisingly, both centromeric clusters and the SC components associated with them persist long after the disassembly of the euchromatic SC at the end of pachytene. The initiation of homologous recombination through the formation of programmed double-strand breaks (DSBs) is not required for either the formation or the maintenance of the centromeric clusters. Our data support a view in which the SC-mediated clustering at the centromeres is the initiating event for meiotic synapsis.
Asunto(s)
Drosophila/genética , Animales , Centrómero/fisiología , Emparejamiento Cromosómico , Segregación Cromosómica , Cromosomas , Drosophila/fisiología , Proteínas de Drosophila/metabolismo , Femenino , Recombinación Homóloga , Oocitos/fisiología , Complejo Sinaptonémico/fisiologíaRESUMEN
Sleep is a fundamental biological process for all animals. However, the molecular mechanisms that regulate sleep are still poorly understood. Here we report that sleep-like behavior in Drosophila is severely impaired by mutations in sarah (sra), a member of the Regulator of Calcineurin (RCAN) family of genes. Sleep reduction in sra mutants is highly correlated with decreases in Sra protein levels. Pan-neural expression of sra rescues this behavioral phenotype, indicating that neuronal sra function is required for normal sleep. Since Sra regulates calcineurin (CN), we generated and examined the behavior of knock-out mutants for all Drosophila CN genes: CanA-14F, Pp2B-14D, and CanA1 (catalytic subunits), and CanB and CanB2 (regulatory subunits). While all mutants show at least minor changes in sleep, CanA-14F(KO) and CanB(KO) have striking reductions, suggesting that these are the major CN subunits regulating sleep. In addition, neuronal expression of constitutively active forms of CN catalytic subunits also significantly reduces sleep, demonstrating that both increases and decreases in CN activity inhibit sleep. sra sleep defects are suppressed by CN mutations, indicating that sra and CN affect sleep through a common mechanism. Our results demonstrate that CN and its regulation by Sra are required for normal sleep in Drosophila and identify a critical role of Ca(2+)/calmodulin-dependent signaling in sleep regulation.
Asunto(s)
Calcineurina/fisiología , Proteínas de Drosophila/fisiología , Drosophila/fisiología , Péptidos y Proteínas de Señalización Intracelular/fisiología , Sueño/fisiología , Animales , Animales Modificados Genéticamente , Western Blotting , Señalización del Calcio/genética , Señalización del Calcio/fisiología , Proteínas de Unión al Calcio , Longevidad/genética , Masculino , Actividad Motora/fisiología , Mutación/fisiología , Plasticidad Neuronal/fisiologíaRESUMEN
Ca(2+) signaling pathways play important roles to complete meiosis from metaphase II arrest in vertebrate oocytes. However, less is known about the molecular mechanism of completion of meiosis in Drosophila females. Here, we provide direct evidence that calcineurin, a Ca(2+)/calmodulin (CaM)-dependent phosphatase, is essential for meiotic progression beyond metaphase I in Drosophila oocytes. Oocytes from germline clones lacking CanB2, a calcineurin regulatory subunit B, failed to complete meiosis after egg activation, and laid eggs exhibited a meiotic arrested anaphase I chromosome configuration. Genetic analyses suggest that calcineurin activity is regulated by Sarah (Sra), a family member of regulators of calcineurin (RCANs), through a Sra phosphorylation-dependent mechanism. Our results support a view in which the phosphorylation of Sra not only acts to relieve the inhibitory effects of Sra, but also acts to activate calcineurin, thus explaining the role of RCAN proteins as positive regulators of calcineurin.
Asunto(s)
Calcineurina/metabolismo , Drosophila/genética , Drosophila/metabolismo , Anafase , Animales , Calcineurina/genética , Cromosomas/metabolismo , Femenino , Fertilización/genética , Meiosis , Metafase , Oocitos/metabolismo , Oocitos/fisiología , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
In Drosophila oocytes achiasmate homologs are faithfully segregated to opposite poles at meiosis I via a process referred to as achiasmate homologous segregation. We observed that achiasmate homologs display dynamic movements on the meiotic spindle during mid-prometaphase. An analysis of living prometaphase oocytes revealed both the rejoining of achiasmate X chromosomes initially located on opposite half-spindles and the separation toward opposite poles of two X chromosomes that were initially located on the same half spindle. When the two achiasmate X chromosomes were positioned on opposite halves of the spindle their kinetochores appeared to display proper co-orientation. However, when both Xs were located on the same half spindle their kinetochores appeared to be oriented in the same direction. Thus, the prometaphase movement of achiasmate chromosomes is a congression-like process in which the two homologs undergo both separation and rejoining events that result in the either loss or establishment of proper kinetochore co-orientation. During this period of dynamic chromosome movement, the achiasmate homologs were connected by heterochromatic threads that can span large distances relative to the length of the developing spindle. Additionally, the passenger complex proteins Incenp and Aurora B appeared to localize to these heterochromatic threads. We propose that these threads assist in the rejoining of homologs and the congression of the migrating achiasmate homologs back to the main chromosomal mass prior to metaphase arrest.
Asunto(s)
Drosophila melanogaster/embriología , Drosophila melanogaster/fisiología , Heterocromatina/química , Oscilometría , Animales , Segregación Cromosómica , Cromosomas/metabolismo , Heterocromatina/metabolismo , Modelos Biológicos , Modelos Genéticos , Oocitos/metabolismo , Prometafase , Huso AcromáticoRESUMEN
Decapentaplegic (Dpp), a Drosophila homologue of bone morphogenetic proteins, acts as a morphogen to regulate patterning along the anterior-posterior axis of the developing wing. Previous studies showed that Dally, a heparan sulfate proteoglycan, regulates both the distribution of Dpp morphogen and cellular responses to Dpp. However, the molecular mechanism by which Dally affects the Dpp morphogen gradient remains to be elucidated. Here, we characterized activity, stability, and gradient formation of a truncated form of Dpp (Dpp(Delta N)), which lacks a short domain at the N-terminus essential for its interaction with Dally. Dpp(Delta N) shows the same signaling activity and protein stability as wild-type Dpp in vitro but has a shorter half-life in vivo, suggesting that Dally stabilizes Dpp in the extracellular matrix. Furthermore, genetic interaction experiments revealed that Dally antagonizes the effect of Thickveins (Tkv; a Dpp type I receptor) on Dpp signaling. Given that Tkv can downregulate Dpp signaling by receptor-mediated endocytosis of Dpp, the ability of dally to antagonize tkv suggests that Dally inhibits this process. Based on these observations, we propose a model in which Dally regulates Dpp distribution and signaling by disrupting receptor-mediated internalization and degradation of the Dpp-receptor complex.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Glicoproteínas de Membrana/metabolismo , Proteoglicanos/metabolismo , Alas de Animales/embriología , Animales , Línea Celular , Proteínas de Drosophila/química , Drosophila melanogaster/metabolismo , Matriz Extracelular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Receptores de Superficie Celular/metabolismo , Alas de Animales/metabolismoRESUMEN
Many meiotic systems in female animals include a lengthy arrest in G2 that separates the end of pachytene from nuclear envelope breakdown (NEB). However, the mechanisms by which a meiotic cell can arrest for long periods of time (decades in human females) have remained a mystery. The Drosophila Matrimony (Mtrm) protein is expressed from the end of pachytene until the completion of meiosis I. Loss-of-function mtrm mutants result in precocious NEB. Coimmunoprecipitation experiments reveal that Mtrm physically interacts with Polo kinase (Polo) in vivo, and multidimensional protein identification technology mass spectrometry analysis reveals that Mtrm binds to Polo with an approximate stoichiometry of 1:1. Mutation of a Polo-Box Domain (PBD) binding site in Mtrm ablates the function of Mtrm and the physical interaction of Mtrm with Polo. The meiotic defects observed in mtrm/+ heterozygotes are fully suppressed by reducing the dose of polo+, demonstrating that Mtrm acts as an inhibitor of Polo. Mtrm acts as a negative regulator of Polo during the later stages of G2 arrest. Indeed, both the repression of Polo expression until stage 11 and the inactivation of newly synthesized Polo by Mtrm until stage 13 play critical roles in maintaining and properly terminating G2 arrest. Our data suggest a model in which the eventual activation of Cdc25 by an excess of Polo at stage 13 triggers NEB and entry into prometaphase.
Asunto(s)
Proteínas de Drosophila/metabolismo , Fase G2 , Meiosis , Proteínas Serina-Treonina Quinasas/metabolismo , Aminoácidos/genética , Aminoácidos/metabolismo , Animales , Animales Modificados Genéticamente , Sitios de Unión , Centrómero/genética , Segregación Cromosómica/genética , Regulación hacia Abajo , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Dosificación de Gen/genética , Regulación del Desarrollo de la Expresión Génica , Heterocigoto , Datos de Secuencia Molecular , Mutación/genética , Membrana Nuclear/metabolismo , Fase Paquiteno , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Huso Acromático/metabolismoRESUMEN
The Drosophila gene ald encodes the fly ortholog of mps1, a conserved kinetochore-associated protein kinase required for the meiotic and mitotic spindle assembly checkpoints. Using live imaging, we demonstrate that oocytes lacking Ald/Mps1 (hereafter referred to as Ald) protein enter anaphase I immediately upon completing spindle formation, in a fashion that does not allow sufficient time for nonexchange homologs to complete their normal partitioning to opposite half spindles. This observation can explain the heightened sensitivity of nonexchange chromosomes to the meiotic effects of hypomorphic ald alleles. In one of the first studies of the female meiotic kinetochore, we show that Ald localizes to the outer edge of meiotic kinetochores after germinal vesicle breakdown, where it is often observed to be extended well away from the chromosomes. Ald also localizes to numerous filaments throughout the oocyte. These filaments, which are not observed in mitotic cells, also contain the outer kinetochore protein kinase Polo, but not the inner kinetochore proteins Incenp or Aurora-B. These filaments polymerize during early germinal vesicle breakdown, perhaps as a means of storing excess outer kinetochore kinases during early embryonic development.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas de Drosophila/genética , Drosophila/citología , Drosophila/genética , Meiosis/genética , Proteínas Quinasas/genética , Anafase/genética , Animales , Proteínas de Ciclo Celular/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Femenino , Genes de Insecto , Cinetocoros/metabolismo , Meiosis/fisiología , Mitosis/genética , Modelos Genéticos , Mutación , Oocitos/citología , Oocitos/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina QuinasasRESUMEN
Modulatory calcineurin-interacting proteins (MCIPs)--also termed regulators of calcineurin (RCNs), calcipressins, or DSCR1 (Down's syndrome critical region 1)--are highly conserved regulators of calcineurin, a Ca(2+)/calmodulin-dependent protein phosphatase . Although overexpression experiments in several organisms have revealed that MCIPs inhibit calcineurin activity , their in vivo functions remain unclear. Here, we show that the Drosophila MCIP sarah (sra) is essential for meiotic progression in oocytes. Eggs from sra null mothers are arrested at anaphase of meiosis I. This phenotype was due to loss of function of sra specifically in the female germline. Sra is physically associated with the catalytic subunit of calcineurin, and its overexpression suppresses the phenotypes caused by constitutively activated calcineurin, such as rough eye or loss of wing veins. Hyperactivation of calcineurin signaling in the germline cells resulted in a meiotic-arrest phenotype, which can also be suppressed by overexpression of Sra. All these results support the hypothesis that Sra regulates female meiosis by controlling calcineurin activity in the germline. To our knowledge, this is the first unambiguous demonstration that the regulation of calcineurin signaling by MCIPs plays a critical role in a defined biological process.
Asunto(s)
Calcineurina/metabolismo , Proteínas de Drosophila/fisiología , Drosophila/metabolismo , Péptidos y Proteínas de Señalización Intracelular/fisiología , Meiosis , Óvulo/citología , Anafase/genética , Animales , Calcineurina/genética , Proteínas de Unión al Calcio , Drosophila/anatomía & histología , Drosophila/citología , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Femenino , Fertilidad/genética , Regulación de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Meiosis/genética , Movimiento/fisiología , Mutación , Ovario/citología , Ovario/metabolismo , Óvulo/metabolismo , Fenotipo , Subunidades de Proteína/metabolismo , Transducción de SeñalRESUMEN
Glypicans, a family of heparan sulfate proteoglycans attached to the cell surface via a glycosylphosphatidylinositol (GPI)-anchor, play essential roles in morphogen signaling and distributions. A Drosophila glypican, Dally, regulates the gradient formation of Decapentaplegic (Dpp) in the developing wing. To gain insights into the function of glypicans in morphogen signaling, we examined the activities of two mutant forms of Dally: a transmembrane form (TM-Dally) and a secreted form (Sec-Dally). Misexpression of tm-dally in the wing disc had a similar yet weaker effect in enhancing Dpp signaling compared to that of wild-type dally. In contrast, Sec-Dally shows a weak dominant negative activity on Dpp signal transduction. Furthermore, sec-dally expression led to patterning defects as well as a substantial overgrowth of tissues and animals through the expansion of the action range of Hh. These findings support the recently proposed model that secreted glypicans have opposing and/or distinct effects on morphogen signaling from the membrane-tethered forms.
Asunto(s)
Tipificación del Cuerpo/fisiología , Proteínas de Drosophila/metabolismo , Regulación del Desarrollo de la Expresión Génica , Glicoproteínas de Membrana/metabolismo , Proteoglicanos/metabolismo , Transducción de Señal/fisiología , Alas de Animales/embriología , Animales , Cartilla de ADN , Drosophila , Proteínas de Drosophila/genética , Proteínas Hedgehog , Immunoblotting , Inmunohistoquímica , Glicoproteínas de Membrana/genética , Proteoglicanos/genéticaRESUMEN
Alzheimer's beta-secretase (BACE1), an aspartic protease, cleaves amyloid precursor protein to produce a neurotoxic peptide, amyloid-beta, which plays a role in triggering Alzheimer's disease. We previously found that BACE1 also cleaves a glycosyltransferase, alpha2,6-sialyltransferase, as a physiological substrate. In the present study, we performed a BLAST homology search, identified two Drosophila aspartic proteases that are homologous to human BACE1, and isolated their cDNAs. The proteins encoded by the cDNAs were designated as DASP1 and DASP2, which exhibited 59% and 50% similarity to human BACE1, respectively. Each protein contained a pair of active site motifs (Asp/Thr or Ser/Gly), which is a common characteristic of aspartic proteases including BACE1. Although DASP1 and DASP2 did not contain an apparent transmenbrane domain, the proteases overexpressed in COS cells were localized in the Golgi area. Some of the DASP1 overexpressed in S2 cells was secreted, but none of the DASP2 was. DASP1 transcripts were expressed in the head of fruitflies, whereas DASP2 transcripts were mainly expressed in the body. When either DASP1 or DASP2 was coexpressed together with a Golgi-resident transferase, Drosophila heparan sulfate 6-O-sulfotransferase, the protease enhanced the secretion of the transferase from the cells, indicating that both DASP1 and DASP2 can induce the secretion of the 6-O-sulfotransferase.
Asunto(s)
Ácido Aspártico Endopeptidasas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimología , Aparato de Golgi/enzimología , Sulfotransferasas/metabolismo , Secuencia de Aminoácidos , Secretasas de la Proteína Precursora del Amiloide , Animales , Ácido Aspártico Endopeptidasas/química , Ácido Aspártico Endopeptidasas/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Endopeptidasas , Genes de Insecto , Aparato de Golgi/metabolismoRESUMEN
To better understand the genetic bases of postmating responses in Drosophila melanogaster females, we screened a collection of P{GS} insertion lines and identified two insertions in sarah (sra), whose misexpression in the nervous system induced high levels of ovulation in virgins. The gene sra encodes a protein similar to human Down syndrome critical region 1 (DSCR1). The ovulation phenotype was reproduced in transgenic virgins expressing UAS-sra in the nervous system. The flies also extruded the ovipositor toward courting males as seen in wild-type mated females, supporting the notion that ovulation and behavioral patterns are physiologically coupled. The sra insertions were found to be hypomorphic alleles with reduced expression levels. Females homozygous for these alleles show: (1) spontaneous ovulation in virgins, (2) sterility with impaired meiotic progression, and (3) compromised postmating responses with lower ovulation level, higher remating rate, and shorter period for restoration of receptivity. No obvious defects were observed in the homozygous males. The gene sra is predominantly expressed in oocytes, nurse cells, and the nervous system. Taken together, these results indicate that the expression level of sra is critical for ovulation and female courtship behavior, including their postmating changes.
Asunto(s)
Drosophila melanogaster/fisiología , Proteínas Musculares/genética , Ovulación/fisiología , Conducta Sexual Animal/fisiología , Secuencia de Aminoácidos , Animales , Encéfalo/metabolismo , Proteínas de Unión al ADN , Drosophila melanogaster/genética , Femenino , Vectores Genéticos , Péptidos y Proteínas de Señalización Intracelular , Datos de Secuencia Molecular , Proteínas Musculares/fisiología , Oogénesis/genética , Oogénesis/fisiologíaRESUMEN
Hyaluronan (HA) is a large linear polymer of repeating disaccharides of glucuronic acid and GlcNAc. Although HA is widely distributed in vertebrate animals, it has not been found in invertebrates, including insect species. Insects utilize chitin, a repeating beta-1,4-linked homopolymer of GlcNAc, as a major component of their exoskeleton. Recent studies illustrate the similarities in the biosynthetic mechanisms of HA and chitin and suggest that HA synthase (HAS) and chitin synthase have evolved from a common ancestral molecule. Although the biochemical properties and in vivo functions of HAS proteins have been extensively studied, the molecular basis for HA biosynthesis is not completely understood. For example, it is currently not clear if proper chain elongation and secretion of HA require other components in addition to HAS. Here, we demonstrate that a non-HA-synthesizing animal, the fruit fly Drosophila melanogaster, can produce HA in vivo when a single HAS protein is introduced. Expression of the mouse HAS2 gene in Drosophila tissues by the Gal4/UAS (upstream activating sequence) system resulted in massive HA accumulation in the extracellular space and caused various morphological defects. These morphological abnormalities were ascribed to disordered cell-cell communications due to accumulation of HA rather than disruption of heparan sulfate synthesis. We also show that adult wings with HA can hold a high level of water. These findings demonstrate that organisms synthesizing chitin (but not HA) are capable of producing HA that is structurally and functionally relevant to that in mammals. The ability of insect cells to produce HA supports the idea that in vivo HA biosynthesis does not require molecules other than the HAS protein. An alternative model is that Drosophila cells use endogenous components of the chitin biosynthetic machinery to produce and secrete HA.